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# Enzyme Kinetics

## Rate of Enzyme Catalysis vs Substrate Concentration

For many enzymes, the rate of
catalysis, V, varies with the
substrate concentration, [S].
V is defined as the number of
moles of product formed per
second.
At a fixed concentration of
enzyme, V is almost linearly
proportional to the initial
increasing concentration of [S].
At very high [S], V is independent
of [S].

## The Michaelis-Menten Approach to Enzyme Kinetics

(Leonor) Michaelis and (Maud) Menten devised a model for the kinetics of enzymecatalyzed reactions.
The model proposed is the one that accounts for the kinetic properties of many
enzymes:

An enzyme, E, combines with S to form an ES complex, with a rate constant k1. The ES
complex has two possible fates. It can dissociate to E and S, with a rate constant of k-1,
or it can proceed to form product, P, with a rate constant k2.
Michaelis and Menten made a derivation of these kinetic characteristics to arrive at the
Michaelis-Menten Equation:

Vinit =

Vmax [S]
KM + [S]

Mi chael is-Menten
equation

## Michaelis-Menten Equation Derivation

For an enzyme-catalyzed reaction
E + S

k1
k-1

ES

k2

## The rates of formation and breakdown of ES are

given by these equations
rate of formation of ES = k 1 [E][S]
rate of breakdown of ES = k

-1 [ES]

k1 [E][S] = k-1[ES] + k 2[ES]

+ k 2[ES]

## Michaelis-Menten Equation Derivation (cont.)

When the steady state is reached, the concentration
of free enzyme is the total less that bound in ES
[E] = [E]T - [ES]

## Substituting for the concentration of free enzyme and

collecting all rate constants in one term gives
([E]T - [ES]) [S]
[ES]

k-1 + k2

= KM

k1

## Michaelis-Menten Equation Derivation (cont.)

It is now possible to solve for the concentration of the
enzyme-substrate complex, [ES]
[E]T [S] - [ES][S]
= KM
[ES]
[E]T [S] - [ES][S] = KM [ES]
[E]T [S] = [ES](K M + [S])

Or alternatively

[E]T [S]
[ES] =
KM + [S]

## Michaelis-Menten Equation Derivation (cont.)

In the initial stages, formation of product depends only on the
rate of breakdown of ES

Vinit = k 2 [ES]

k 2[E]T [S]
=
KM + [S]

## If substrate concentration is so large that the enzyme is

saturated with substrate [ES] = [E]T

## V init = V max = k 2 [E]T

Substituting k2[E]T = Vmax into the top equation gives

Vinit =

Vmax [S]
KM + [S]

Mi chael is-Menten
equation

## The Meaning of KM or the Michaelis constant

Initial concentration of substrate at Vmax or at half saturation of E
with S
Ratio of the constants for the breakdown of ES to constants of its
[E]T [S] - [ES][S]
formation:
= KM
[ES]
[E]T [S] - [ES][S] = KM [ES]

## A measure of the affinity of E for S: if k2 is <k-1 or k1, neglecting k2,

[E]T [S] = [ES](K M + [S])
therefore KM = k-1/k1
The lower the KM value, the more affinity has the enzyme for its
substrate

The [S]=
Meaning
KM orreduces
the Michaelis
constant
When
KM, the of
equation
to

V=

Vmax [S]
KM + [S]

Vmax [S]
[S] + [S]

Vmax
2

## Linearizing The Michaelis-Menten Equation

It is difficult to determine Vmax experimentally
Vmax
[S]
The equation
of
Michaelis
and Menten
is for
V=
(an
equation
fora ahyperbola
hyperbola)

KM + [S]

## The equation can be transformed into the equation for a

straight line by taking the reciprocal of each side

KM + [S]
Vmax [S]

KM
+
=
Vmax [S]

1
V

1
V

KM
+ 1
=
Vmax [S]
Vmax

[S]
Vmax [S]

Lineweaver-Burk Plot
1 form
1 + b, and is the
The Lineweaver-Burke
the
y = mx
1 = KMplot has
+

## formula forVa straight

line
Vmax
[S]
V
max

a plot of 1/V versus 1/[S] will give a straight line with slope of
KM/Vmax and y intercept of 1/Vmax
such a plot is known as a Lineweaver-Burk double reciprocal
plot
Double reciprocal plots can easily be understood and provide
recognizable pattern for the study of ENZYME INHIBITION

KLineweaver-Burk
M is the
dissociation
constant for ES; the
greater the value of
KM, the less tightly S
is bound to E

## Vmax is the turnover

number

Plot (Contd)

Turnover Numbers
Vmax is related to the turnover number of enzyme:also
called kmax
cat

[ET ]

## Number of moles of substrate that react to form product

per mole of enzyme per unit of time

Enzyme Inhibition
Reversible inhibitor: a substance that binds to an enzyme to inhibit it,
but can be released

## competitive inhibitor: binds to the active (catalytic) site and

noncompetitive inhibitor: binds to a site other than the active
site; inhibits the enzyme by changing its conformation
Irreversible inhibitor: a substance that causes inhibition that cannot be
reversed

## usually involves formation or breaking of covalent bonds to or

on the enzyme

Competitive Inhibition
Substrate must compete with inhibitor for the active site;
more substrate is required to reach a given reaction
velocity
EI
I + E + S
ES
P

## We can write a dissociation constant, KI for EI

EI

KI =

[E][I]
[EI]

Competitive Inhibition

Competitive
Inhibition
No inhibiti
on
y =

KM
Vmax
m

1
V

1
S

1
Vmax
b

1
V

KM
Vmax

1 +

[I]
KI

1
S

1
Vmax
b

## In a Lineweaver-Burk double reciprocal plot of 1/V versus 1/[S], the

slope (and the x intercept) changes but the y intercept does not
change

## Noncompetitive Inhibition (Contd)

+S
Several equilibria are involved
E

-I

ES

-S
+I

-I
+S

EI

+I

ESI

-S

V

max

Vmax
1 + [I]/KI

E + P

## A Lineweaver-Burke Plot for Noncompetitive

Inhibition
Because the inhibitor does not interfere with binding of
substrate to the active site, KM is unchanged
Increasing substrate concentration cannot overcome
noncompetitive
inhibition
No inhibiti
on
1 = KM 1 + 1
V
S
Vmax
Vmax
y =
m x + b
In the presence of a noncompetitive inhibitor

1
V

KM
Vmax

1 +
m

[I]
KI

1
S

+
+

1
Vmax

1 +
b

[I]
KI

## A Lineweaver-Burke Plot for Noncompetitive

Inhibition (Contd)

## Other Types of Inhibition

Uncompetitive- inhibitor can bind to the ES complex but not to free
E. Vmax decreases and KM decreases.

## Mixed- Similar to noncompetitively, but binding of I affects binding of

S and vice versa.

Uncompetitive Inhibition

Uncompetitive Inhibition

Summary
Effects Type
of reversible
inhibitors on kinetic
constants
of Inhibitor
Effect
Competitive (I binds to E only)